BMP-15 compositions

ABSTRACT

Purified BMP-15-related proteins and processes for producing them are disclosed. DNA molecules encoding the BMP-15-related proteins are also disclosed. The proteins may be used in the treatment of bone and cartilage and/or other connective tissue defects and in wound healing and related tissue repair.

This application is a continuation of U.S. Ser. No. 08/798,665, filedFeb. 11, 1997 and now, which application is a divisional of U.S. Ser.No. 08/446,924, filed May 18, 1995 and now issued as U.S. Pat. No.5,635,372.

The present invention relates to a novel family of purified proteinsdesignated BMP-15 and BMP-15-related proteins, DNA encoding them, andprocesses for obtaining them. These proteins may be used to induce boneand/or cartilage or other connective tissue formation, and in woundhealing and tissue repair. These proteins may also be used foraugmenting the activity of other bone morphogenetic proteins.

BACKGROUND OF THE INVENTION

The search for the molecule or molecules responsible for the bone-,cartilage-, and other connective tissue-inductive activity present inbone and other tissue extracts has led to the discovery of a novel setof molecules called the Bone Morphogenetic Proteins (BMPs). Thestructures of several proteins, designated BMP-1 through BMP-14, havepreviously been elucidated. The unique inductive activities of theseproteins, along with their presence in bone, suggests that they areimportant regulators of bone repair processes, and may be involved inthe normal maintenance of bone tissue. There is a need to identifywhether additional proteins exist which play a role in these processes.The present invention relates to the identification of such a protein,which the inventors have designated BMP-15.

SUMMARY OF THE INVENTION

As used herein, the term "BMP-15-related protein" refers to the humanBMP-15 protein, having the amino acid sequence specified in SEQUENCE IDNO:4, as well as homologues of this protein found in other species; andother proteins which are closely related structurally and/orfunctionally to BMP-15. Examples of "BMP-15-related proteins" includemurine PC-3 protein, having the amino acid sequence of SEQUENCE ID NO:2,as well as homologues in other species, particularly human.

Murine PC-3

The murine PC-3 DNA sequence (SEQ ID NO:1) and amino acid sequence (SEQID NO: 2) are set forth in the Sequence Listings. BMP-15-relatedproteins are capable of inducing the formation of cartilage, bone, orother connective tissue, or combinations thereof. PC-3 proteins may befurther characterized by the ability to demonstrate cartilage and/orbone and/or other connective tissue formation activity in the rat boneformation assay described below.

Murine PC-3 may be produced by culturing a cell transformed with a DNAsequence comprising nucleotide a DNA sequence encoding the mature PC-3polypeptide, comprising nucleotide #634 to nucleotide #1008 as shown inSEQ ID NO: 1, and recovering and purifying from the culture medium aprotein characterized by the amino acid sequence comprising amino acids#1 to #125 as shown in SEQ ID NO:2 substantially free from otherproteinaceous materials with which it is co-produced. For production inmammalian cells, the DNA sequence further comprises a DNA sequenceencoding a suitable propeptide 5' to and linked in frame to thenucleotide sequence encoding the mature PC-3 polypeptide. The propeptidemay be the native PC-3 propeptide, or may be a propeptide from anotherprotein of the TGF-β superfamily.

It is expected that other species, particularly human, have DNAsequences homologous to murine PC-3. The invention, therefore, includesmethods for obtaining the DNA sequences encoding human PC-3, the DNAsequences obtained by those methods, and the human protein encoded bythose DNA sequences. This method entails utilizing the murine PC-3nucleotide sequence or portions thereof to design probes to screenlibraries for the human gene or coding sequences or fragments thereofusing standard techniques. Thus, the present invention includes DNAsequences from other species, particularly, human, which are homologousto murine PC-3 and can be obtained using the murine PC-3 sequence. A DNAsequence encoding the complete mature human BMP-15 protein (SEQ ID NO:3)and the corresponding amino acid sequence (SEQ ID NO:4) are set forthherein. As described herein, these sequences were isolated using aportion of the murine PC-3 sequence as a probe. The human BMP-15sequence of SEQUENCE ID NO:3 may also be used in order to design probesto obtain the complete human BMP-15 gene or coding sequences throughstandard techniques. The murine PC-3 and human BMP-15 sequences, orportions thereof, may also be used as probes, or to design probes, inorder to obtain other related DNA sequences. The BMP-15-related proteinsof the present invention, such as human BMP-15, may be produced byculturing a cell transformed with the correlating DNA sequence, such asthe BMP-15 DNA sequence, and recovering and purifying protein, such asBMP-15, from the culture medium. The purified expressed protein issubstantially free from other proteinaceous materials with which it isco-produced, as well as from other contaminants. The recovered purifiedprotein is contemplated to exhibit cartilage and/or bone and/orconnective tissue formation activity. The proteins of the invention maybe further characterized by the ability to demonstrate cartilage and/orbone and/or other connective tissue formation activity in the rat boneformation assay described below.

Another aspect of the invention provides pharmaceutical compositionscontaining a therapeutically effective amount of a BMP-15-relatedprotein, such as murine or human PC-3 or BMP-15 protein, in apharmaceutically acceptable vehicle or carrier. These compositions ofthe invention may be used in the formation of bone. These compositionsmay further be utilized for the formation of cartilage, or otherconnective tissue, including tendon, ligament, meniscus and otherconnective tissue, as well as combinations of the above, for exampleregeneration of the tendon-to-bone attachment apparatus. Thecompositions of the present invention, such as compositions of humanBMP-15, may also be used for wound healing and tissue repair.Compositions of the invention may further include at least one othertherapeutically useful agent such as the BMP proteins BMP-1, BMP-2,BMP-3, BMP4, BMP-5, BMP-6 and BMP-7, disclosed for instance in U.S. Pat.Nos. 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and5,141,905; BMP-8, disclosed in PCT publication WO91/18098; and BMP-9,disclosed in PCT publication WO93/00432, BMP-10, disclosed in PCTapplication WO94/26893; BMP-11, disclosed in PCT application WO94/26892,or BMP-12 or BMP-13, disclosed in co-pending patent application, Ser.No. 08/362,670, filed on Dec. 22, 1994. Other compositions which mayalso be useful include Vgr-2, and any of the GDFs, including thosedescribed in PCT applications WO94/15965; WO94/15949; WO95/01801;WO95/01802; WO94/21681; WO94/15966; and others. Also useful in thepresent invention may be BIP, disclosed in WO94/01557; and MP52,disclosed in PCT application WO93/16099. The disclosures of all of theabove applications are hereby incorporated by reference.

The compositions of the invention may comprise, in addition to aBMP-15-related protein, other therapeutically useful agents includinggrowth factors such as epidermal growth factor (EGF), fibroblast growthfactor (FGF), transforming growth factor (TGF-α and TGF-β), activins,inhibins, and insulin-like growth factor (IGF). The compositions mayalso include an appropriate matrix for instance, for supporting thecomposition and providing a surface for bone and/or cartilage and/orother connective tissue growth. The matrix may provide slow release ofthe osteoinductive protein and/or the appropriate environment forpresentation thereof.

The BMP-15-related compositions may be employed in methods for treatinga number of bone and/or cartilage and/or other connective tissuedefects, periodontal disease and healing of various types of tissues andwounds. The tissue and wounds which may be treated include epidermis,nerve, muscle, including cardiac muscle, and other tissues and wounds.These methods, according to the invention, entail administering to apatient needing such bone and/or cartilage and/or other connectivetissue formation, wound healing or tissue repair, an effective amount ofa BMP-15-related protein. The BMP-15-related compositions may also beused to treat or prevent such conditions as osteoarthritis,osteoporosis, and other abnormalities of bone, cartilage or otherconnective tissue and other tissues. These methods may also entail theadministration of a protein of the invention in conjunction with atleast one other BMP protein as described above. In addition, thesemethods may also include the administration of a BMP-15-related proteinwith other growth factors including EGF, FGF, TGF-α, TGF-β, activin,inhibin and IGF.

Still a further aspect of the invention are DNA sequences coding forexpression of a BMP-15-related protein. Such sequences include thesequence of nucleotides in a 5' to 3' direction illustrated in SEQ IDNO: 3, DNA sequences which, but for the degeneracy of the genetic code,are identical to the DNA sequence SEQ ID NO: 3, and encode the proteinof SEQ ID NO: 4. Further included in the present invention are DNAsequences which hybridize under stringent conditions with the DNAsequence of SEQ ID NO: 3 and encode a protein having the ability toinduce the formation of cartilage and/or bone and/or other connectivetissue. Preferred DNA sequences include those which hybridize understringent conditions [see, T. Maniatis et al, Molecular Cloning (ALaboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to389]. It is generally preferred that such DNA sequences encode apolypeptide which is at least about 80% homologous, and more preferablyat least about 90% homologous, to the mature human BMP-15 amino acidsequence shown in SEQ ID NO:4. Finally, allelic or other variations ofthe sequences of SEQ ID NO: 3, whether such nucleotide changes result inchanges in the peptide sequence or not, but where the peptide sequencestill has BMP-15 activity, are also included in the present invention.

A further aspect of the invention includes vectors comprising a DNAsequence as described above in operative association with an expressioncontrol sequence therefor. These vectors may be employed in a novelprocess for producing a BMP-15-related protein of the invention in whicha cell line transformed with a DNA sequence encoding a BMP-15-relatedprotein in operative association with an expression control sequencetherefor, is cultured in a suitable culture medium and a BMP-15-relatedprotein is recovered and purified therefrom. This process may employ anumber of Icnown cells both prokaryotic and eukaryotic as host cells forexpression of the polypeptide. The vectors may be used in gene therapyapplications. In such use, the vectors may be transfected into the cellsof a patient in vitro, and the cells may be reintroduced into a patient.Alternatively, the vectors may be introduced into a patient in vivothrough targeted transfection.

The purified proteins of the present inventions may be used to generateantibodies, either monoclonal or polyclonal, to human BMP-15 and/orother BMP-15-related proteins, using methods that are known in the artof antibody production. Thus, the present invention also includesantibodies to human BMP-15 and/or other BMP-15 related proteins. Theantibodies may be useful for purification of BMP-15 and/or other BMP-15related proteins, or for inhibiting or preventing the effects of BMP-15related proteins. The proteins or compositions of the present inventionmay also be useful for treating cell populations, such as embryoniccells or stem cell populations, to enhance or enrich the growth and/ordifferentiation of the cells. The treated cell populations may be usefulfor gene therapy applications.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleotide sequence encoding the entire mature murinePC-3.

SEQ ID NO:2 is the amino acid sequence containing the mature murine PC-3polypeptide.

SEQ ID NO:3 is a nucleotide sequence encoding the entire mature humanBMP- 15.

SEQ ID NO:4 is the amino acid sequence containing the mature humanBMP-15 polypeptide.

SEQ ID NO:5 is a consensus sequences of members of the BMP/TGF-β/Vg-1family of proteins; wherein the first Xaa is either Gin, Asn or Asp; thesecond Xaa is either Asp, Glu or Asn; and the third Xaa is either Val orIle.

SEQ ID NO:6 is primer #1, directed to the consensus sequence of SEQ IDNO:5.

SEQ ID NO:7 is a consensus sequence of members of the BMP/TGF-β/Vg-1family of proteins; wherein the Xaa is either Val or Leu.

SEQ ID NO:8 is primer #2, directed to the consensus sequences of SEQ IDNO:7.

SEQ ID NO: 9 is the nucleotide sequence of a fragment of murine PC-3isolated using the primers of SEQ ID NO: 6 and SEQ ID NO:8.

SEQ ID NO: 10 is the derived amino acid sequence encoded by thenucleotide sequence of SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide sequence of oligonucleotide primer #3,directed to the murine PC-3 DNA sequence, used to isolate a full-lengthmPC-3 gene.

SEQ ID NO: 12 is the nucleotide sequence of oligonucleotide primer #4,directed to the murine PC-3 DNA sequence, used to isolate a full-lengthmPC-3 gene.

SEQ ID NO:13 is the nucleotide sequence of oligonucleotide primer #5,directed to the murine PC-3 DNA sequence, used to isolate a full-lengthhuman BMP-15 gene.

SEQ ID NO:14 is the nucleotide sequence of oligonucleotide primer #6,directed to the murine PC-3 DNA sequence, used to isolate a full lengthhuman BMP-15 gene.

DETAILED DESCRIPTION OF THE INVENTION

BMP-15

The murine PC-3 nucleotide sequence (SEQ ID NO: 1) and encoded aminoacid sequence (SEQ ID NO: 2) are set forth in the Sequence listingsherein. The coding sequence of the mature murine PC-3 protein begins atnucleotide #634 and continues through nucleotide #1008. Purified murinePC-3 proteins of the present invention are produced by culturing a hostcell transformed with a DNA sequence comprising the DNA coding sequenceof SEQ ID NO: 1 from nucleotide #634 to #1008, or from nucleotide #490to #1008, and recovering and purifying from the culture medium a proteinwhich contains the amino acid sequence or a substantially homologoussequence as represented by amino acids #-48 to #125 or #1 to #125 of SEQID NO: 2.

The human BMP-15 sequence of the present invention is obtained using thewhole or fragments of the murine PC-3 DNA sequence, or a partial humanBMP-15 sequence, as a probe. Thus, the human BMP-15 DNA sequencecomprise the DNA sequence of nucleotides #1002 to #1376 of SEQ ID NO: 3.This sequence of the human BMP-15 DNA sequence corresponds well tonucleotides #634 to #1008 of the murine PC-3 DNA sequence shown in SEQID NO: 1. The human BMP-15 protein comprises the sequence of amino acids#1 to #125 of SEQ ID NO: 4.

It is expected that BMP-15 protein, as expressed by mammalian cells suchas CHO cells, exists as a heterogeneous population of active species ofBMP-15 protein with varying N-termini. It is expected that activespecies will comprise an amino acid sequence beginning with the cysteineresidue at amino acid #24 of SEQ ID NO:4, or will comprise additionalamino acid sequence further in the N-terminal direction. Thus, it isexpected that DNA sequences encoding active BMP-15 proteins willcomprise a nucleotide sequence comprising nucleotides #576, #813, #1002or #1071 to #1373 or #1376 of SEQ ID NO: 3. Accordingly, active speciesof human BMP-15 are expected to include those comprising amino acids#-142, #-63, #1 or #24 to #124 or #125 of SEQ ID NO:4.

A host cell may be transformed with a coding sequence encoding apropeptide suitable for the secretion of proteins by the host cell islinked in proper reading frame to the coding sequence for the maturePC-3 or BMP-15 protein. For example, see U.S. Pat. No. 5,168,050, thedisclosure of which is hereby incorporated by reference, in which a DNAencoding a precursor portion of a mammalian protein other than BMP-2 isfused to the DNA encoding a mature BMP-2 protein. See also thespecification of co-pending patent application, Ser. No. 08/362,670,filed on Dec. 22, 1994, in which the propeptide of BMP-2 is fused to theDNA encoding a mature BMP-12 protein. The disclosure of both of thesereferences are hereby incorporated by reference. Thus, the presentinvention includes chimeric DNA molecules comprising a DNA sequenceencoding a propeptide from a member of the TGF-β superfamily ofproteins, other than BMP-15, is linked in correct reading frame to a DNAsequence encoding a BMP-15-related protein, such as PC-3 or BMP-15protein. The term "chimeric" is used to signify that the propeptideoriginates from a different polypeptide than the BMP-15-related protein.

The N-terminus of one active species of human BMP- 15 is expected to beexperimentally determined by expression in E. coli to be as follows:[M]QADGISAE. Thus, it appears that the N-terminus of this species ofBMP-15 is at amino acid #1 of SEQ ID NO: 3, and a DNA sequence encodingsaid species of BMP-15 would comprise nucleotides #1002 to #1376 of SEQID NO: 3. The apparent molecular weight of human BMP-15 monomer isexpected to be experimentally determined by SDS-PAGE to be approximately10-17 kd on a Novex 16% tricine gel. The human BMP-15 protein isexpected to exist as a clear, colorless solution in 0.1% trifluoroaceticacid.

It is expected that other BMP-15-related proteins, such as PC-3, asexpressed by mammalian cells such as CHO cells, also exist as aheterogeneous population of active species of BMP-15-related proteinwith varying N-termini. For example, it is expected that active speciesof PC-3 will comprise an amino acid sequence beginning with the cysteineresidue at amino acid #24 of SEQ ID NO:2, or will comprise additionalamino acid sequence further in the N-terminal direction. Thus, it isexpected that DNA sequences encoding active PC-3 proteins include thosewhich comprise a nucleotide sequence comprising nucleotides #427, #490,#634, #640, #664 or #703 to #1005 or #1008 of SEQ ID NO: 1. Accordingly,active PC-3 proteins include those comprising amino acids #-69, #-48,#1, #3, #11 or #24 to #124 or #125.

The BMP-15-related proteins of the present invention, includepolypeptides having a molecular weight of about 10-17 kd in monomericform, said polypeptide comprising the amino acid sequence of SEQ IDNO:10 and having the ability to induce the formation of cartilage and/orbone and/or other connective tissue in the Rosen-Modified Sampath-Reddiectopic implant assay, described in the examples.

The BMP-15-related proteins recovered from the culture medium arepurified by isolating them from other proteinaceous materials from whichthey are co-produced and from other contaminants present. BMP-15-relatedproteins may be characterized by the ability to induce the formation ofcartilage and/or bone and/or other connective tissue, for example, inthe rat bone formation assay described below.

The BMP-15-related proteins provided herein also include factors encodedby the sequences similar to those of SEQ ID NO:1 or SEQ ID NO:3, butinto which modifications are naturally provided (e.g. allelic variationsin the nucleotide sequence which may result in amino acid changes in thepolypeptide) or deliberately engineered. For example, syntheticpolypeptides may wholly or partially duplicate continuous sequences ofthe amino acid residues of SEQ ID NO:2 or SEQ ID NO:4. These sequences,by virtue of sharing primary, secondary, or tertiary structural andconformational characteristics with bone growth factor polypeptides ofSEQ ID NO: 2 or SEQ ID NO: 4 may possess bone growth factor biologicalproperties in common therewith. Thus, they may be employed asbiologically active substitutes for naturally-occurring BMP-15 and otherBMP-15-related polypeptides in therapeutic processes.

Other specific mutations of the sequences of BMP-15-related proteinsdescribed herein involve modifications of glycosylation sites. Thesemodifications may involve O-linked or N-linked glycosylation sites. Forinstance, the absence of glycosylation or only partial glycosylationresults from amino acid substitution or deletion at asparagine-linkedglycosylation recognition sites. The asparagine-linked glycosylationrecognition sites comprise tripeptide sequences which are specificallyrecognized by appropriate cellular glycosylation enzymes. Thesetripeptide sequences are either asparagine-X-threonine orasparagine-X-serine, where X is usually any amino acid. A variety ofamino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Additionally, bacterial expressionof BMP-15-related protein will also result in production of anon-glycosylated protein, even if the glycosylation sites are leftunmodified.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding for expression of BMP-15-related proteins. These DNAsequences include those depicted in SEQ ID NO: 1 and SEQ ID NO: 3 in a5' to 3' direction and those sequences which hybridize thereto understringent hybridization conditions [for example, 0.1×SSC, 0.1% SDS at65° C.; see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual),Cold Spring Harbor Laboratory (1982), pages 387 to 389] and encode aprotein having cartilage and/or bone and/or other connective tissueinducing activity. These DNA sequences also include those which comprisethe DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and those whichhybridize thereto under stringent hybridization conditions and encode aprotein having cartilage and/or bone and/or other connective tissueinducing activity.

Similarly, DNA sequences which code for BMP-15-related proteins codedfor by the sequences of SEQ ID NO: 1 or SEQ ID NO: 3, or BMP-15-relatedproteins which comprise the amino acid sequence of SEQ ID NO: 2 or SEQID NO: 4, but which differ in codon sequence due to the degeneracies ofthe genetic code or allelic variations (naturally-occurring base changesin the species population which may or may not result in an amino acidchange) also encode the novel factors described herein. Variations inthe DNA sequences of SEQ ID NO: 1 or SEQ ID NO: 3 which are caused bypoint mutations or by induced modifications (including insertion,deletion, and substitution) to enhance the activity, half-life orproduction of the polypeptides encoded are also encompassed in theinvention.

Another aspect of the present invention provides a novel method forproducing BMP-15-related proteins. The method of the present inventioninvolves culturing a suitable cell line, which has been transformed witha DNA sequence encoding a BMP-15-related protein of the invention, underthe control of known regulatory sequences. The transformed host cellsare cultured and the BMP-15-related proteins recovered and purified fromthe culture medium. The purified proteins are substantially free fromother proteins with which they are co-produced as well as from othercontaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). The selection of suitable mammalian hostcells and methods for transformation, culture, amplification, screening,product production and purification are known in the art. See, e.g.,Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively,Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al,U.S. Pat. No. 4,419,446. Another suitable mammalian cell line, which isdescribed in the accompanying examples, is the monkey COS-1 cell line.The mammalian cell CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method. Forexpression of the protein in bacterial cells, DNA encoding thepropeptide of BMP-15-related is generally not necessary.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of these novel BMP-15-related polypeptides.Preferably the vectors contain the full novel DNA sequences describedabove which encode the novel factors of the invention. Additionally, thevectors contain appropriate expression control sequences permittingexpression of the BMP-15-related protein sequences. Alternatively,vectors incorporating modified sequences as described above are alsoembodiments of the present invention. Additionally, the sequence of SEQID NO:1, SEQ ID NO:3 or other sequences encoding BMP-15-related proteinscould be manipulated to express a mature BMP-15-related protein bydeleting BMP-15-related propeptide sequences and replacing them withsequences encoding the complete propeptides of other BMP proteins ormembers of the TGF-, superfamily. Thus, the present invention includeschimeric DNA molecules encoding a propeptide from a member of the TGF-0superfamily linked in correct reading frame to a DNA sequence encoding aBMP-15-related polypeptide.

The vectors may be employed in the method of transforming cell lines andcontain selected regulatory sequences in operative association with theDNA coding sequences of the invention which are capable of directing thereplication and expression thereof in selected host cells. Regulatorysequences for such vectors are known to those skilled in the art and maybe selected depending upon the host cells. Such selection is routine anddoes not form part of the present invention.

A protein of the present invention, which induces cartilage and/or boneand/or other connective tissue formation in circumstances where suchtissue is not normally formed, has application in the healing of bonefractures and cartilage or other connective tissue defects in humans andother animals. Such a preparation employing a BMP-15-related protein mayhave prophylactic use in closed as well as open fracture reduction andalso in the improved fixation of artificial joints. De novo boneformation induced by an osteogenic agent contributes to the repair ofcongenital, trauma induced, or oncologic resection induced craniofacialdefects, and also is useful in cosmetic plastic surgery. ABMP-15-related protein may be used in the treatment of periodontaldisease, and in other tooth repair processes. Such agents may provide anenvironment to attract bone-forming cells stimulate, growth ofbone-forming cells or induce differentiation of progenitors ofbone-forming cells, and may also support the regeneration of theperiodontal ligament and attachment apparatus, which connects bone andteeth. BMP-15-related polypeptides of the invention may also be usefulin the treatment of osteoporosis. A variety of osteogenic,cartilage-inducing and bone inducing factors have been described. See,e.g., European patent applications 148,155 and 169,016 for discussionsthereof.

The proteins of the invention may also be used in wound healing andrelated tissue repair. The types of wounds include, but are not limitedto burns, incisions and ulcers. (See, e.g. PCT Publication WO84/01106for discussion of wound healing and related tissue repair). It isfurther contemplated that proteins of the invention may increaseneuronal survival and therefore be useful in transplantation andtreatment of conditions exhibiting a decrease in neuronal survival. Theproteins of the invention may further be useful for the treatment ofconditions related to other types of tissue, such as epidermis andmuscle.

A further aspect of the invention is a therapeutic method andcomposition for repairing fractures and other conditions related tocartilage and/or bone and/or other connective tissue defects orperiodontal diseases. The invention further comprises therapeuticmethods and compositions for wound healing and tissue repair. Suchcompositions comprise a therapeutically effective amount of at least oneof the BMP-15-related proteins of the invention in admixture with apharmaceutically acceptable vehicle, carrier or matrix. It is furthercontemplated that compositions of the invention may increase neuronalsurvival and therefore be useful in transplantation and treatment ofconditions exhibiting a decrease in neuronal survival. Compositions ofthe invention may further include at least one other therapeuticallyuseful agent, such as members of the TGF-β superfamily of proteins,which includes the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6and BMP-7, disclosed for instance in U.S. Pat. No. 5,108,922; 5,013,649;5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCTpublication WO91/18098; BMP-9, disclosed in PCT publication WO93/00432;BMP-10, disclosed in PCT application WO94/26893; BMP-11, disclosed inPCT application WO94/26892, or BMP-12 or BMP-13, disclosed in co-pendingpatent application, Ser. No. 08/362,670, U.S. Pat. No. 5,658,882 filedon Dec. 22, 1994. Other compositions which may also be useful includeVgr-2, and any of the GDFs, including those described in PCTapplications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681;WO94/15966; and others. Also useful in the present invention may be BIP,disclosed in WO94/01557; and MP52, disclosed in PCT applicationWO93/16099. The disclosures of the above applications are herebyincorporated by reference herein.

It is expected that BMP-15 and BMP-15-related proteins may exist innature as homodimers or heterodimers. To promote the formation of dimersof BMP-15 and BMP-15-related proteins with increased stability, one cangenetically engineer the DNA sequence of SEQUENCE ID NO:1 or SEQUENCE IDNO:3 to provide one or more additional cysteine residues to increasepotential dimer formation. The resulting DNA sequence would be capableof producing a "cysteine added variant" of BMP-15 or a BMP-15 relatedprotein. In a preferred embodiment, one would engineer the DNA sequenceof SEQUENCE ID NO:3 so that the codon appearing at nucleotides #1266 to#1268 is altered to a nucleotide triplet encoding a cysteine residue,such as TGT or TGC. Similarly, one could alter the DNA sequence ofSEQUENCE ID NO:1 to replace the codon triplet at nucleotides #898 to#900 to a triplet encoding a cysteine residue, such as TGT or TGC.Alternatively, one can produce "cysteine added variants" of BMP-15 orBMP-15-related proteins by altering the sequence of the protein at theamino acid level, for example, by altering the amino acid sequences ofSEQUENCE ID NO:2 or SEQUENCE ID NO:4 at amino acid residue #89 from Serto Cys. Production of "cysteine added variants" of proteins is describedin U.S. Pat. No. 5,166,322, the disclosure of which is herebyincorporated by reference.

It is expected that the proteins of the invention may act in concertwith or perhaps synergistically with other related proteins and growthfactors. Further therapeutic methods and compositions of the inventiontherefore comprise a therapeutic amount of at least one BMP-15-relatedprotein of the invention with a therapeutic amount of at least one othermember of the TGF-β superfamily of proteins, such as the BMP proteinsdisclosed in the applications described above. Such combinations maycomprise separate molecules of the BMP proteins or heteromoleculescomprised of different BMP moieties. For example, a method andcomposition of the invention may comprise a disulfide linked dimercomprising a BMP-15-related protein subunit and a subunit from one ofthe "BMP" proteins described above. Thus, the present invention includesa purified BMP-15-related polypeptide which is a heterodirner whereinone subunit comprises the amino acid sequence from amino acid #1 toamino acid #125 of SEQ ID NO:2 or amino acid #1 to #125 of SEQ ID NO:4,and one subunit comprises an amino acid sequence for a bonemorphogenetic protein selected from the group consisting of BMP-1,BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11or BMP-12 or BMP-13, disclosed in co-pending patent application, No.WO95/16035, filed on Dec. 22, 1994. A further embodiment may comprise aheterodimer of BMP-15-related moieties, for example of human BMP-15 andthe human homologue of murine PC-3. Further, BMP-15-related proteins maybe combined with other agents beneficial to the treatment of the boneand/or cartilage and/or other connective tissue defect, wound, or tissuein question. These agents include various growth factors such asepidermal growth factor (EGF), fibroblast growth factor (FGF), plateletderived growth factor (PDGF), transforming growth factors (TGF-A andTGF-0), activins, inhibins, and k-fibroblast growth factor (kFGF),parathyroid hormone (PTH), leukemia inhibitory factor (LIF/HILDA/DIA),insulin-like growth factors (IGF-I and IGF-II). Portions of these agentsmay also be used in compositions of the present invention.

The preparation and formulation of such physiologically acceptableprotein compositions, having due regard to pH, isotonicity, stabilityand the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the lack of species specificity in BMP proteins. Particularlydomestic animals and thoroughbred horses in addition to humans aredesired patients for such treatment with the BMP-15-related proteins ofthe present invention. The therapeutic method includes administering thecomposition topically, systemically, or locally as an implant or device.When administered, the therapeutic composition for use in this inventionis, of course, in a pyrogen-free, physiologically acceptable form.Further, the composition may desirably be encapsulated or injected in aviscous form for delivery to the site of bone, cartilage or otherconnective tissue or other tissue damage. Topical administration may besuitable for wound healing and tissue repair. Therapeutically usefulagents other than the BMP-15-related proteins which may also optionallybe included in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with theBMP composition in the methods of the invention.

Preferably for bone and/or cartilage and/or other connective tissueformation, the composition includes a matrix capable of deliveringBMP-15-related or other BMP proteins to the site of bone and/orcartilage and/or other connective tissue damage, providing a structurefor the developing bone and cartilage and other connective tissue andoptimally capable of being resorbed into the body. The matrix mayprovide slow release of BMP-15-related and/or other bone inductiveprotein, as well as proper presentation and appropriate environment forcellular infiltration. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the BMP-15-relatedcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalciumphosphate. The bioceramics may be altered incomposition, such as in calcium-aluminate-phosphate and processing toalter pore size, particle size, particle shape, and biodegradability.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of theBMP-15-related protein, e.g. amount of bone weight desired to be formed,the site of bone damage, the condition of the damaged bone, the size ofa wound, type of damaged tissue, the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and the types of BMP proteins in the composition. Theaddition of other known growth factors, such as IGF I (insulin likegrowth factor I), to the final composition, may also effect the dosage.

Progress can be monitored by periodic assessment of bone growth and/orrepair. The progress can be monitored, for example, x-rays,histomorphometric determinations and tetracycline labeling.

The following examples illustrate practice of the present invention inrecovering and characterizing murine PC-3 protein and employing the DNAit to recover human BMP-15 and other BMP-15-related proteins, obtainingthe human proteins and expressing the proteins via recombinanttechniques.

EXAMPLES Example 1

Isolation of DNA

DNA sequences encoding BMP-15 and BMP-15 related proteins, such as themurine PC-3 protein may be isolated by various techniques known to thoseskilled in the art. As described below, oligonucleotide primers may bedesigned on the basis of amino acid sequences present in other BMPproteins, Vg-1 related proteins and other proteins of the TGF-βsuperfamily. Regions containing amino acid sequences which are highlyconserved within the BMP family of proteins and within other members ofthe TGF-β superfamily of proteins can be identified and consensus aminoacid sequences of these highly conserved regions can be constructedbased on the similarity of the corresponding regions of individualBMP/TGF-β/Vg-1 proteins. An example of such a consensus amino acidsequence is indicated below.

Consensus Amino Acid Sequence (1):

Trp-Gln/Asn/Asp-Asp/Glu/Asn-Trp-Ile-Val/Ile-Ala (SEQ ID NO: #5) WhereX/Y indicates that either amino acid residue may appear at thatposition.

The following oligonucleotide is designed on the basis of the aboveidentified consensus amino acid sequence (1):

#1: GCGGATCCTGGVANGABTGGATHRTNGC (SEQ ID NO:#6)

This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The standard nucleotide symbols in the above identifiedoligonucleotide primer are as follows: A,adenosine; C,cytosine;G,guanine; T,thymine; N,adenosine or cytosine or guanine or thymine;R,adenosine or cytosine; Y,cytosine or thymine; H,adenosine or cytosineor thymine; V,adenosine or cytosine or guanine; D,adenosine or guanineor thymine.

The first eight nucleotides of oligonucleotide #1 (underlined) containthe recognition sequence for the restriction endonuclease BamHI in orderto facilitate the manipulation of a specifically amplified DNA sequenceencoding the PC-3 protein and are thus not derived from the consensusamino acid sequence (1) presented above.

A second consensus amino acid sequence is derived from another highlyconserved region of BMP/TGF-β/Vg-1 proteins as described below:

Asn-His-Ala-Ile-Val/Leu-Gln-Thr (SEQ ID NO:#7)

The following oligonucleotide is designed on the basis of the aboveidentified consensus amino acid sequence (2):

#2: GCTCTAGAGTYTGNAYNATNGCRTGRTT (SEQ ID NO:8)

This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The same nucleotide symbols are used as described above.

The first eight nucleotides of oligonucleotide #2 (underlined) containthe recognition sequence for the restriction endonuclease XbaI in orderto facilitate the manipulation of a specifically amplified DNA sequenceencoding the PC-3 protein and are thus not derived from the consensusamino acid sequence (2) presented above.

It is contemplated that the PC-3 protein of the invention and otherBMP/TGF-β/Vg-1 related proteins may contain amino acid sequences similarto the consensus amino acid sequences described above and that thelocation of those sequences within a BMP-15 or PC-3 protein or othernovel related proteins would correspond to the relative locations in theproteins from which they were derived. It is further contemplated thatthis positional information derived from the structure of otherBMP/TGF-β/Vg-1 proteins and the oligonucleotide sequences #1 and #2which have been derived from consensus amino acid sequences (1) and (2),respectively, could be utilized to specifically amplify DNA sequencesencoding the corresponding amino acids of a BMP-15 protein or otherBMP/TGF-β/Vg-1 related proteins.

Based on the knowledge of the gene structures of BMP/TGF-β/Vg-1proteins, it is further contemplated that human or murine genomic DNAcan be used as a template to perform specific amplification reactionswhich would result in the identification of BMP-15 BMP/TGF-β/Vg-1(BMP-15 related protein) encoding sequences. Such specific amplificationreactions of a human or murine genomic DNA template could be initiatedwith the use of oligonucleotide primers #1 and #2 described earlier.Oligonucleotides #1 and #2 identified above are utilized as primers toallow the specific amplification of a specific nucleotide sequence frommurine genomic DNA. The amplification reaction is performed as follows:

Murine genomic DNA is sheared by repeated passage through a 25 gaugeneedle, denatured at 100° C. for 5 minutes and then chilled on icebefore adding to a reaction mixture containing 200 μM eachdeoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP), 10 mMTris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 1.25 units TaqDNA polymerase, 50 pM oligonucleotide #1 and 50 pM oligonucleotide #2,in a total reaction volume of 50 μl. This reaction mixture is subjectedto thermal cycling in the following manner: 1 minute at 94° C., 1 minuteat 37° C., 2 minutes at 72° C. for thirty cycles; followed by a 7 minuteincubation at 72° C.

The DNA which is specifically amplified by this reaction is ethanolprecipitated, digested with the restriction endonucleases BamHI and XbaIand subjected to agarose gel electrophoresis. A region of the gel,corresponding to the predicted size of the BMP-15 or otherBMP/TGF-β/Vg-1 encoding DNA fragment, is excised and the specificallyamplified DNA fragments contained therein are electroeluted andsubcloned into the plasmid vector pGEM-3 between the XbaI and BamHIsites of the polylinker. DNA sequence analysis of one of the resultingBMP-15 related subclones indicates the specifically amplified DNAsequence product contained therein encodes a portion of theBMP-15-related protein, mPC-3, of the invention.

The DNA sequence (SEQ ID NO:9) and derived amino acid sequence (SEQ IDNO:10) of this specifically amplified DNA fragment of mPC-3 are shown inthe SEQUENCE Listings.

Nucleotides #1-#26 of SEQ ID NO:9 comprise a portion of oligonucleotide#1 and nucleotides #100-#119 comprise a portion of the reversecompliment of oligonucleotide #2 utilized to perform the specificamplification reaction. Due to the function of oligonucleotides #1 and#2 in initiating the amplification reaction, they may not correspondexactly to the actual sequence encoding a PC-3 protein and are thereforenot translated in the corresponding amino acid derivation (SEQ IDNO:10).

The following oligonucleotide probes are designed on the basis of thespecifically amplified PC-3 murine DNA sequence set forth above (SEQ IDNO:9) and synthesized on an automated DNA synthesizer:

#3: TCCTCGTCTCTATACCCCAAATTACTGTAAAGGAATCTGT(SEQ ID NO:11) and

#4: ATCTGTACTCGGGTATTACCCTATGGTCTCAATTCACCC (SEQ ID NO:#12)

Oligonucleotide probes #3 and #4 are designed on the basis ofnucleotides #27-#66 and #61-#99 of the murine PC-3 sequence set forth inSEQ ID NO:9.

These oligonucleotide probes are radioactively labeled with ³² P andemployed to screen a murine genomic library constructed in the vectorλFIX II (Stratagene catalog #946309). 500,000 recombinants of the humangenomic library are plated at a density of approximately 10,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques are made one set ofnitrocellulose filters is hybridized to oligonucleotide probe #3 and theduplicate set of nitrocellulose filters is hybridized to oligonucleotide#4, both in a hybridization buffer consisting of 5×SSC, 1% SDS, 10%dextran sulfate, 2×Denhardt's , 100 μg/ml herring sperm DNA) at 60° C.overnight. The following day the radioactively labelled oligonucleotidecontaining hybridization solution is removed an the filters are washedwith 5×SSC, 0.1% SDS at 60° C. Two recombinants which hybridize to botholigonucleotide probes are identified and one is plaque purified. Thisplaque purified recombinant bacteriophage clone which hybridizes to themurine PC-3 oligonucleotide probes #3 and #4 is designated .Oslashed.60. A bacteriophage plate stock is made and bacteriophage DNA isisolated from the .O slashed.60 murine genomic clone. The completeinsert of the .O slashed.60 murine genomic recombinant is excised withthe restriction endonuclease NotI, subcloned into a plasmid vector(pBluescript) and DNA sequence analysis is performed. This plasmidsubclone is designated mPC-3/NotI-18 and has been deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md."ATCC" under the accession #69777 on Mar. 30, 1995. This deposit meetsthe requirements of the Budapest Treaty of the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure andRegulations thereunder.

The partial DNA sequence (SEQ ID NO:1) and derived amino acid sequence(SEQ ID NO:2) of the approximately 18 kb DNA insert of the plasmidsubclone mPC-3/NotI-18, derived from clone .O slashed.60, are shown inthe Sequence Listings.

It should be noted that nucleotides #765-#837 of SEQ ID NO:1 correspondto nucleotides #28-#99 of the specifically amplified murine PC-3encoding DNA fragment set forth in SEQ ID NO:9 thus confirming that themurine genomic bacteriophage clone .O slashed.60 and derivative subclonemPC-3/NotI-18 encode at least a portion of the PC-3 protein of theinvention. The nucleotide sequence of a portion of the 18 kb NotI insertof the plasmid mPC-3/NotI-18 contains an open reading frame of 885 basepairs, as defined by nucleotides #124-#1008 of SEQ ID NO:1.

The 5' limit of this open reading frame is defined by a stop codon atnucleotide positions #121-#123. As this sequence is derived from agenomic clone it is difficult to determine the boundary between the 5'extent of coding sequence and the 3' limit of intervening sequence(intron/non-coding sequence), however, nucleotides #127-#154 arepredicted to represent sequences characteristic of a splice acceptorsite, including the invariant dinucleotide AG found at positions#153-#154, which delineate the 3' limit of an intron in genomicsequences. This predicts the 5' boundary of a single exon which encodesthe entire mature peptide and a substantial portion of the propeptide ofthe murine PC-3 protein of the invention. The 852 base pair open readingframe of this predicted exon (nucleotides #157-#1008 of SEQ ID NO:1)encodes at least 284 amino acids of the murine PC-3 protein of theinvention. The encoded 284 amino acid murine PC-3 protein includes thefull mature murine PC-3 peptide (amino acids #1-#125 of SEQ ID NO:2), aswell as the C-terminal portion of the propeptide region of the primarytranslation product (amino acid #-159 to #-1 of SEQ ID NO:2).

Based on the knowledge of other BMP proteins and other proteins withinthe TGF-β family, it is predicted that the precursor polypeptide wouldbe cleaved at the multibasic sequence Arg-Ser-Val-Arg (amino acids-470-1 of SEQ ID NO: 2) in agreement with a proposed consensusproteolytic processing sequence of Arg-Xaa-Xaa-Arg. Cleavage of themurine PC-3 precursor polypeptide is expected to generate a 125 aminoacid mature peptide beginning with the amino acid Gin at position #1 ofSEQ ID NO:2. The processing of murine PC-3 into the mature form isexpected to involve dimerization and removal of the N-terminal region ina manner analogous to the processing of the related protein TGF-β[Gentry et al., Molec & Cell. Biol., 8:4162 (1988); Derynck et al.Nature, 316:701 (1985)].

It is contemplated therefore that the mature active species of murinePC-3 comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #125 of SEQ ID NO:1 with a predictedmolecular weight of approximately 14,000 daltons. Further active speciesare contemplated comprising at least amino acids #24 to #125 of SEQ IDNO:2, thereby including the first conserved cysteine residue.

As with other members of the TGF-β/BMP family of proteins, thecarboxy-terminal portion of the murine PC-3 protein exhibits greatersequence conservation than the more amino-terminal portion. The percentamino acid identity of the murine PC-3 protein in the cysteine-richC-terminal domain (amino acids #24-#125) to the corresponding region ofhuman BMP proteins and other proteins within the TGF-β family is asfollows: BMP-2, 42%; BMP-3, 39%; BMP-4, 41%; BMP-5, 39%; BMP-6, 40%;BMP-7, 38%; BMP-8, 35%; BMP-9, 38%; BMP-10, 40%; BMP-11, 33%; Vgl, 39%;GDF-1, 32%; TGF-β1, 26%; TGF-β2, 30%; TGF-β3, 29%; inhibin α(B), 30%;inhibin α(A), 34%.

The murine PC-3 DNA sequence (SEQ ID NO:1), or a portion thereof, can beused as a probe to identify a human cell line or tissue whichsynthesizes PC-3 or PC-3-related MRNA. Briefly described, RNA isextracted from a selected cell or tissue source and eitherelectrophoresed on a formaldehyde agarose gel and transferred tonitrocellulose, or reacted with formaldehyde and spotted onnitrocellulose directly. The nitrocellulose is then hybridized to aprobe derived from the coding sequence of murine PC-3.

Alternatively, the murine PC-3 sequence is used to designoligonucleotide primers which will specifically amplify a portion of thePC-3 or PC-3-related encoding sequence located in the region between theprimers utilized to perform the specific amplification reaction. It iscontemplated that these murine PC-3 derived primers would allow one tospecifically amplify corresponding human PC-3 or PC-3-related encodingsequences from mRNA, cDNA or genomic DNA templates. Once a positivesource has been identified by one of the above described methods, mRNAis selected by oligo (dT) cellulose chromatography and cDNA issynthesized and cloned in λgt10 or other λ bacteriophage vectors knownto those skilled in the art, for example, λZAP by established techniques(Toole et al., supra). It is also possible to perform theoligonucleotide primer directed amplification reaction, described above,directly on a pre-established human cDNA or genomic library which hasbeen cloned into a λ bacteriophage vector. In such cases, a librarywhich yields a specifically amplified DNA product encoding a portion ofthe human PC-3 or PC-3-related protein could be screened directly,utilizing the fragment of amplified human PC-3 or PC-3-related proteinencoding DNA as a probe.

Oligonucleotide primers designed on the basis of the DNA sequence of themurine PC-3 genomic clone .O slashed.60 are predicted to allow thespecific amplification of human PC-3 or PC-3-related encoding DNAsequences from pre-established human cDNA libraries which arecommercially available (i.e., Stratagene, La Jolla, Calif. or ClonetechLaboratories, Inc., Palo Alto, Calif.). The following oligonucleotideprimer is designed on the basis of nucleotides #728 to #747 of the DNAsequence set forth in SEQ ID NO:1 and synthesized on an automated DNAsynthesizer:

#5: GCTTCCACCAACTAGGCTGG (SEQ ID NO:13)

The following oligonucleotide primer is designed on the basis of thereverse compliment of nucleotides #1007-#988 of the DNA sequence setforth in SEQ ID NO:1 and synthesized on an automated DNA synthesizer:

#6: CTACATGTACAGGACTGGGC (SEQ ID NO:14)

The standard nucleotide symbols in the above identified primers are asfollows: A, adenine; C, cytosine; G, guanine; T, thymine.

Primers #5 and #6 identified above are utilized as primers to allow theamplification of a specific PC-3 or PC-3-related encoding nucleotidesequence from pre-established cDNA libraries.

Approximately 1×10⁸ pfu (plaque forming units) of λbacteriophagelibraries containing human cDNA inserts such as those detailed above aredenatured at 95° C. for five minutes prior to addition to a reactionmixture containing 200 μM each deoxynucleotide triphosphates (dATP,dGTP, dCTP and dTTP) 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.001% gelatin, 1.25 units Taq DNA polymerase, 100 pM oligonucleotideprimer #4 and 100 pM oligonucleotide primer #5. The reaction mixture isthen subjected to thermal cycling in the following manner: 1 minute at94° C., 1 minute at 50° C., 1 minute at 72° C. for thirty-nine cyclesfollowed by 10 minutes at 72° C.

The DNA which is specifically amplified by this reaction would beexpected to generate a PC-3 or PC-3-related protein-encoding product ofapproximately 280 base pairs. The resulting 280 bp DNA product isvisualized following electrophoresis of the reaction products through a2% agarose gel. Once a positive cDNA source has been identified in thismanner, the corresponding cDNA library from which a PC-3 specific orPC-3-related sequence was amplified could be screened directly with the280 bp insert or other PC-3 specific probes in order to identify andisolate cDNA clones encoding the full-length PC-3 or PC-3-relatedprotein of the invention.

Additional methods known to those skilled in the art may be used toisolate other full-length cDNAs encoding human PC-3 related proteins, orfull length cDNA clones encoding PC-3 related proteins of the inventionfrom species other than humans, particularly other mammalian species.

Alternatively, oligonucleotides #5 and #6 described above (SEQ ID NO: 13and SEQ ID NO:14) are utilized as primers to allow the specificamplification of murine PC-3 specific nucleotide sequences from murinePC-3 encoding plasmid mPC-3/NotI-18. The amplification reaction isperformed as follows: Approximately 25 ng of mPC-3/NotI-18 plasmid DNAis added to a reaction mixture containing 200 μM each deoxynucleotidetriphosphates (dATP, dGTP, dCTP and dTTP) 10 mM Tris-HCl pH 8.3, 50 mMKCl, 1.5 mM MgCl₂, 0.001% gelatin, 1.25 units Taq DNA polymerase, 100 pMoligonucleotide primer #5 and 100 pM oligonucleotide primer #6. Thereaction mixture is then subjected to thermal cycling in the followingmanner: 1 minute at 94° C., 1 minute at 53° C., 1 minute at 72° C. forthirty cycles.

The DNA which is specifically amplified by this reaction would beexpected to generate a PC-3 or PC-3-related encoding product ofapproximately 280 base pairs. The resulting 280 bp DNA product isvisualized following electrophoresis of the reaction products through a2% agarose gel. The region of the gel containing the 280 base pairmurine PC-3 DNA fragment is excised and the specifically amplified DNAfragments contained therein are extracted (by electroelution or by othermethods known to those skilled in the art). The gel-extracted 280 basepair DNA amplification product was radioactively labelled with ³² P andemployed to screen a human genomic library constructed in the vector λDASH II (Stratagene catalog #945203).

Human BMP-15

One million recombinants of the human genomic library are plated at adensity of approximately 20,000 recombinants per plate on 50 plates.Duplicate nitrocellulose replicas of the recombinant bacteriophageplaques are hybridized, under reduced stringency conditions, to thespecifically amplified 280 bp probe in standard hybridization buffer(SHB=533 SSC, 0.1% SDS, 5×Denhardt's , 100 μg/ml salmon sperm DNA) at60° C. overnight. The following day the radioactively labelledoligonucleotide containing hybridization solution is removed an thefilters are washed, under reduced stringency conditions, with 2×SSC,0.1% SDS at 60° C. Multiple positively hybridizing recombinants areidentified and plaque purified. One of the recombinant bacteriophageclones which hybridizes to the 280 base pair mPC-3 probe is designatedλJLDc19. This recombinant bacteriophage clone is plaque purified, abacteriophage plate stock is made and bacteriophage DNA is isolated fromthe λJLDc19 human genomic clone. The bacteriophage λJLDc19 has beendeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 "ATCC" under the accession #97106 onMar. 30, 1995. This deposit meets the requirements of the BudapestTreaty of the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and Regulations thereunder.

The hybridizing region of this recombinant, λJLDc19, is localized to a 3kb Eco RI fragment. This fragment is subcloned into a plasmid vector(pGEM-3) and DNA sequence analysis is performed. This plasmid subcloneis designated pGEMJLDc19/3.0 and has been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209 "ATCC" under the accession #69779 on Mar. 30, 1995. Thisdeposit meets the requirements of the Budapest Treaty of theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder.

The partial DNA sequence (SEQ ID NO:3) and derived amino acid sequence(SEQ ID NO:4) of a portion of the 3.0 kb DNA insert of the plasmidsubclone pGEMJLDc19/3.0, derived from clone λJLDc19, are shown in theSequence Listings

The DNA sequence of a portion of the 3.0 kb EcoRI insert of the plasmidpGEMJLDc19/3.0 is set forth in SEQ ID NO:3, contains an contains an openreading frame of 888 base pairs, as defined by nucleotides #489-#1376 ofSEQ ID NO:4.

The 5' limit of this open reading frame is defined by a stop codon atnucleotide positions #486-#488. As this sequence is derived from agenomic clone it is difficult to determine the boundary between the 5'extent of coding sequence and the 3' limit of intervening sequence(intron/non-coding sequence), however, nucleotides #498-#528 arepredicted to represent sequences characteristic of a splice acceptorsite, including the invariant dinucleotide AG found at positions#527-#528, which delineate the 3' limit of an intron in genomicsequences. This predicts the 5' boundary of a single exon which encodesthe entire mature peptide and a substantial portion of the propeptide ofthe human PC-3-related protein of the invention, designated BMP-15. The846 base pair open reading frame of this predicted exon (nucleotides#531-#1376 of SEQ ID NO:3) encodes at least 282 amino acids of the humanBMP-15 protein of the invention. The encoded 282 amino acid human BMP-15protein includes the full mature human BMP-15 peptide (amino acids#1-#125 of SEQ ID NO:4), as well as the C-terminal portion of thepropeptide region of the primary translation product (amino acid #-157to #1 of SEQ ID NO:4).

Based on the knowledge of other BMP proteins and other proteins withinthe TGF-β family, it is predicted that the precursor polypeptide wouldbe cleaved at the multibasic sequence Arg-Arg-Thr-Arg (animo acids -4 to-1 of SEQ ID NO:4) in agreement with a proposed consensus proteolyticprocessing sequence of Arg-Xaa-Xaa-Arg. Cleavage of the human BMP-15precursor polypeptide is expected to generate a 125 amino acid maturepeptide beginning with the amino acid Gln at position #1 of SEQ ID NO:4.The processing of human BMP-15 into the mature form is expected toinvolve dimerization and removal of the N-terminal region in a manneranalogous to the processing of the related protein TGF-β [Gentry et al.,Molec & Cell. Biol., 8:4162 (1988); Derynck et al. Nature, 316:701(1985)].

It is contemplated therefore that the mature active species of humanBMP-15 comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #125 of SEQ ID NO:4 with 2 predictedmolecular weight of approximately 14,000 daltons. Further active speciesare contemplated comprising at least amino acids #24 to #125 of SEQ IDNO:4, thereby including the first conserved cysteine residue. As withother members of the TGF-β/BMP family of proteins, the carboxy-terminalportion of the human BMP-15 protein exhibits greater sequenceconservation than the more amino-terminal portion. The percent aminoacid identity of the human BMP-15 protein in the cysteine-richC-terminal domain (amino acids #24-#125) to the corresponding region ofhuman BMP proteins and other proteins within the TGF-β family is asfollows: BMP-2, 43%; BMP-3, 35%; BMP-4, 42%; BMP-5, 41%; BMP-6, 41%;BMP-7, 39%; BMP-8, 34%; BMP-9, 40%; BMP-10, 43%; BMP-11, 32%; Vgl, 39%;GDF-1, 35%; TGF-β1, 28%; TGF-β2, 30%; TGF-β3, 31%; inhibin α(B), 31%;inhibin α(A), 33%.

Example 2

W-20 BIOASSAYS

A. Description of W-20 cells

Use of the W-20 bone marrow stromal cells as an indicator cell line isbased upon the conversion of these cells to osteoblast-like cells aftertreatment with a BMP protein [Thies et al, Journal of Bone and MineralResearch, 5:305 (1990); and Thies et al, Endocrinology, 130:1318(1992)]. Specifically, W-20 cells are a clonal bone marrow stromal cellline derived from adult mice by researchers in the laboratory of Dr. D.Nathan, Children's Hospital, Boston, Mass. Treatment of W-20 cells withcertain BMP proteins results in (1) increased alkaline phosphataseproduction, (2) induction of PTH stimulated cAMP, and (3) induction ofosteocalcin synthesis by the cells. While (1) and (2) representcharacteristics associated with the osteoblast phenotype, the ability tosynthesize osteocalcin is a phenotypic property only displayed by matureosteoblasts. Furthermore, to date we have observed conversion of W-20stromal cells to osteoblast-like cells only upon treatment with BMPs. Inthis manner, the in vitro activities displayed by BMP treated W-20 cellscorrelate with the in vivo bone forming activity known for BMPs.

Below two in vitro assays useful in comparison of BMP activities ofnovel osteoinductive molecules are described.

B. W-20 Alkaline Phosphatase Assay Protocol

W-20 cells are plated into 96 well tissue culture plates at a density of10,000 cells per well in 200 μl of media (DME with 10% heat inactivatedfetal calf serum, 2 mM glutamine and 100 Units/ml penicillin +100 μg/mlstreptomycin. The cells are allowed to attach overnight in a 95% air, 5%CO₂ incubator at 37° C.

The 200 μl of media is removed from each well with a multichannelpipettor and replaced with an equal volume of test sample delivered inDME with 10% heat inactivated fetal calf serum, 2 mM glutamine and 1%penicillin-streptomycin. Test substances are assayed in triplicate.

The test samples and standards are allowed a 24 hour incubation periodwith the W-20 indicator cells. After the 24 hours, plates are removedfrom the 37° C. incubator and the test media are removed from the cells.

The W-20 cell layers are washed 3 times with 200 μl per well ofcalcium/magnesium free phosphate buffered saline and these washes arediscarded.

50 μl of glass distilled water is added to each well and the assayplates are then placed on a dry ice/ethanol bath for quick freezing.Once frozen, the assay plates are removed from the dry ice/ethanol bathand thawed at 37° C. This step is repeated 2 more times for a total of 3freeze-thaw procedures. Once complete, the membrane bound alkalinephosphatase is available for measurement.

50 μl of assay mix (50 mM glycine, 0.05% Triton X-100, 4 mM MgCl₂, 5 mMp-nitrophenol phosphate, pH=10.3) is added to each assay well and theassay plates are then incubated for 30 minutes at 37° C. in a shakingwaterbath at 60 oscillations per minute.

At the end of the 30 minute incubation, the reaction is stopped byadding 100 μl of 0.2 N NaOH to each well and placing the assay plates onice.

The spectrophotometric absorbance for each well is read at a wavelengthof 405 nanometers. These values are then compared to known standards togive an estimate of the alkaline phosphatase activity in each sample.For example, using known amounts of p-nitrophenol phosphate, absorbancevalues are generated. This is shown in Table I.

                  TABLE I                                                         ______________________________________                                        Absorbance Values for Known Standards                                          of P-Nitrophenol Phosphate                                                     P-nitrophenol phosphate umoles                                                                     Mean absorbance (405 nm)                               ______________________________________                                        0.000              0                                                            0.006 0.261 +/- .024                                                          0.012 0.521 +/- .031                                                          0.018 0.797 +/- .063                                                          0.024 1.074 +/- .061                                                          0.030 1.305 +/- .083                                                        ______________________________________                                    

Absorbance values for known amounts of BMPs can be determined andconverted to μmoles of p-nitrophenol phosphate cleaved per unit time asshown in Table II.

                  TABLE II                                                        ______________________________________                                        Alkaline Phosphatase Values for W-20 Cells                                      Treating with BMP-2                                                           BMP-2 concentration                                                                            Absorbance Reading                                                                         umoles substrate                                ng/ml 405 nmeters per hour                                                  ______________________________________                                        0              0.645        0.024                                               1.56 0.696 0.026                                                              3.12 0.765 0.029                                                              6.25 0.923 0.036                                                              12.50 1.121 0.044                                                             25.0 1.457 0.058                                                              50.0 1.662 0.067                                                              100.0 1.977 0.080                                                           ______________________________________                                    

These values are then used to compare the activities of known amounts ofBMP-15 to BMP-2.

C. Osteocalcin RIA Protocol

W-20 cells are plated at 10⁶ cells per well in 24 well multiwell tissueculture dishes in 2 mls of DME containing 10% heat inactivated fetalcalf serum, 2 mM glutamine. The cells are allowed to attach overnight inan atmosphere of 95% air 5% CO₂ at 37° C.

The next day the medium is changed to DME containing 10% fetal calfserum, 2 mM glutamine and the test substance in a total volume of 2 ml.Each test substance is administered to triplicate wells. The testsubstances are incubated with the W-20 cells for a total of 96 hourswith replacement at 48 hours by the same test medias.

At the end of 96 hours, 50 μl of the test media is removed from eachwell and assayed for osteocalcin production using a radioimmunoassay formouse osteocalcin. The details of the assay are described in the kitmanufactured by Biomedical Technologies Inc., 378 Page Street,Stoughton, Mass. 02072. Reagents for the assay are found as productnumbers BT-431 (mouse osteocalcin standard), BT-432 (goat anti-mouseOsteocalcin), BT-431R (iodinated mouse osteocalcin), BT-415 (normal goatserum) and BT-414 (donkey anti goat IgG). The RIA for osteocalcinsynthesized by W-20 cells in response to BMP treatment is carried out asdescribed in the protocol provided by the manufacturer.

The values obtained for the test samples are compared to values forknown standards of mouse osteocalcin and to the amount of osteocalcinproduced by W-20 cells in response challenge with known amounts ofBMP-2. The values for BMP-2 induce osteocalcin synthesis by W-20 cellsis shown in Table III.

                  TABLE III                                                       ______________________________________                                        Osteocalcin Synthesis by W-20 Cells                                               BMP-2 Concentration ng/ml                                                                      Osteocalcin Synthesis ng/well                            ______________________________________                                        0                0.8                                                            2 0.9                                                                         4 0.8                                                                         8 2.2                                                                         16 2.7                                                                        31 3.2                                                                        62 5.1                                                                        125 6.5                                                                       250 8.2                                                                       500 9.4                                                                       1000 10.0                                                                   ______________________________________                                    

Example 3

ROSEN MODIFIED SAMPTH-REDDI ASSAY

A modified version of the rat bone formation assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. USA, 80:6591-6595 (1983) is used toevaluate bone and/or cartilage and/or other connective tissue activityof BMP proteins. This modified assay in herein called the Rosen-modifiedSampath-Reddi assay. The ethenol precipitation step of the Sampath-Reddiprocedure is replaced by dialyzing (if the composition is a solution) ordiafiltering (if the composition is a suspension) the fraction to beassayed against water. The solution or suspension is then equilibrate to0.1% TFA. The resulting solution is added to 20 mg of rat matrix. A mockrat matrix sample not treated with the protein serves as a control. Thismaterial is frozen and lyophilized and the resulting powder enclosed in#5 gelatin capsules. The capsules are implanted subcutaneously in theabdominal thoracic area of 21-49 day old male Long Evans rats. Theimplants are removed after 7-14 days. Half of each implant is used foralkaline phosphatase analysis [see, Reddi et al, Proc. Natl. Acad. Sci.,69:1601 (1972)].

The other half of each implant is fixed and processed for histologicalanalysis. 1 μm glycolmethacrylate sections are stained with Von Kossaand acid fuschin to score the amount of induced bone and cartilage andother connective tissue formation present in each implant. The terms +1through +5 represent the area of each histological section of an implantoccupied by new bone and/or cartilage cells and matrix. A score of +5indicates that greater than 50% of the implant is new bone and/orcartilage produced as a direct result of protein in the implant. A scoreof +4, +3, +2, and +1 would indicate that greater than 40%, 30%, 20% and10% respectively of the implant contains new cartilage and/or bone.

Alternatively, the implants are inspected for the appearance of tissueresembling embryonic tendon, which is easily recognized by the presenceof dense bundles of fibroblasts oriented in the same plane and packedtightly together. [Tendon/ligament-like tissue is described, forexample, in Ham and Cormack, Histology (JB Lippincott Co. (1979). pp.367-369, the disclosure of which is hereby incorporated by reference].These findings may be reproduced in additional assays in whichtendon/ligament-like tissues are observed in the BMP-15-related proteincontaining implants.

The BMP-15-related proteins of this invention may be assessed foractivity on this assay.

Example 4

Expression of BMP-15

In order to produce murine, human or other mammalian BMP-15-relatedproteins, the DNA encoding it is transferred into an appropriateexpression vector and introduced into mammalian cells or other preferredeukaryotic or prokaryotic hosts by conventional genetic engineeringtechniques. The preferred expression system for biologically activerecombinant human BMP-15 is contemplated to be stably transformedmammalian cells.

One skilled in the art can construct mammalian expression vectors byemploying the sequence of SEQ ID NO: 1 or SEQ ID NO:3, or other DNAsequences encoding BMP-15-related proteins or other modified sequencesand known vectors, such as pCD [Okayama et al., Mol. Cell Biol.,2:161-170 (1982)], pJL3, pJL4 [Gough et al., EMBO J., 4:645-653 (1985)]and pMT2 CXM.

The mammalian expression vector pMT2 CXM is a derivative of p91023(b)(Wong et al., Science 228:810-815, 1985) differing from the latter inthat it contains the ampicillin resistance gene in place of thetetracycline resistance gene and further contains a XhoI site forinsertion of cDNA clones. The functional elements of pMT2 CXM have beendescribed (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693)and include the adenovirus VA genes, the SV40 origin of replicationincluding the 72 bp enhancer, the adenovirus major late promoterincluding a 5' splice site and the majority of the adenovirus tripartiteleader sequence present on adenovirus late mRNAs, a 3' splice acceptorsite, a DHFR insert, the SV40 early polyadenylation site (SV40), andpBR322 sequences needed for propagation in E. coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which hasbeen deposited with the American Type Culture Collection (ATCC),Rockville, Md. (USA) under accession number ATCC 67122. EcoRI digestionexcises the cDNA insert present in pMT2-VWF, yielding pMT2 in linearform which can be ligated and used to transform E. coli HB 101 or DH-5to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts the following sequence:

5' PO-CATGGGCAGCTCGAG-3' (SEQ ID NO: 15)

at nucleotide 1145. This sequence contains the recognition site for therestriction endonuclease Xho I. A derivative of pMT2CXM, termed pMT23,contains recognition sites for the restriction endonucleases PstI, EcoRI, SalI and XhoI. Plasmid pMT2 CXM and pMT23 DNA may be prepared byconventional methods.

pEMC2β1 derived from pMT21 may also be suitable in practice of theinvention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. Asdescribed above EcoRI digestion excises the cDNA insert present inpMT-VWF, yielding pMT2 in linear form which can be ligated and used totransform E. Coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2DNA can be prepared by conventional methods.

pMT21 is derived from pMT2 through the following two modifications.First, 76 bp of the 5' untranslated region of the DHFR cDNA including astretch of 19 G residues from G/C tailing for cDNA cloning is deleted.In this process, a XhoI site is inserted to obtain the followingsequence immediately upstream from

    DHFR:                                                                             5' -CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3'                                                              (SEQ ID NO:16)                                                        PstI       Eco RI XhoI                               

Second, a unique ClaI site is introduced by digestion with EcoRV andXbaI, treatment with Klenow fragment of DNA polymerase I, and ligationto a ClaI linker (CATCGATG). This deletes a 250 bp segment from theadenovirus associated RNA (VAI) region but does not interfere with VAIRNA gene expression or function. pMT21 is digested with EcoRI and XhoI,and used to derive the vector pEMC2B1.

A portion of the EMCV leader is obtained from pMT2-ECAT1 [(S. K. Jung,et al, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI and PstI,resulting in a 2752 bp fragment. This fragment is digested with TaqIyielding an Eco RI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5' TaqI protruding end and a3' XhoI protruding end which has the following sequence:

    5'-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT                                                        (SEQ ID NO:17)                                   TaqI                                                                        -    GAAAAACACGATTGC-3'                                                                  XhoI                                                        

This sequence matches the EMC virus leader sequence from nucleotide 763to 827. It also changes the ATG at position 10 within the EMC virusleader to an ATT and is followed by a XhoI site. A three way ligation ofthe pMT21 Eco RI-XhoI fragment, the EMC virus EcoRI-TaqI fragment, andthe 68 bp oligonucleotide adapter TaqI-XhoI adapter resulting in thevector pEMC2β1.

This vector contains the SV40 origin of replication and enhancer, theadenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

The construction of vectors may involve modification of theBMP-15-related DNA sequences. For instance, BMP-15 cDNA can be modifiedby removing the non-coding nucleotides on the 5' and 3' ends of thecoding region. The deleted non-coding nucleotides may or may not bereplaced by other sequences known to be beneficial for expression. Thesevectors are transformed into appropriate host cells for expression ofBMP-15-related proteins. Additionally, the sequence of SEQ ID NO:1 orSEQ ID NO: 3 or other sequences encoding BMP-15-related proteins can bemanipulated to express a mature BMP-15-related protein by deletingBMP-15 encoding propeptide sequences and replacing them with sequencesencoding the complete propeptides of other BMP proteins.

One skilled in the art can manipulate the sequences of SEQ ID NO: 1 orSEQ ID NO:3 by eliminating or replacing the mammalian regulatorysequences flanking the coding sequence with bacterial sequences tocreate bacterial vectors for intracellular or extracellular expressionby bacterial cells. For example, the coding sequences could be furthermanipulated (e.g. ligated to other known linkers or modified by deletingnon-coding sequences therefrom or altering nucleotides therein by otherknown techniques). The modified BMP-15-related coding sequence couldthen be inserted into a known bacterial vector using procedures such asdescribed in T. Taniguchi et al., Proc. Natl Acad. Sci. USA,77:5230-5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells and a BMP-15-related proteinexpressed thereby. For a strategy for producing extracellular expressionof BMP-15-related proteins in bacterial cells, see, e.g. European patentapplication EPA 177,343.

Similar manipulations can be performed for the construction of an insectvector [See, e.g. procedures described in published European patentapplication 155,476] for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. [See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a BMP-15-related protein of theinvention in mammalian cells may involve the construction of cellscontaining multiple copies of the heterologous BMP-15-related gene. Theheterologous gene is linked to an amplifiable marker, e.g. thedihydrofolate reductase (DHFR) gene for which cells containing increasedgene copies can be selected for propagation in increasing concentrationsof methotrexate (MTX) according to the procedures of Kaufman and Sharp,J. Mol. Biol., 159:601-629 (1982). This approach can be employed with anumber of different cell types.

For example, a plasmid containing a DNA sequence for a BMP-15-relatedprotein of the invention in operative association with other plasmidsequences enabling expression thereof and the DHFR expression plasmidpAdA26SV(A)3 [Kaufman and Sharp, Mol. Cell. Biol., 2:1304 (1982)] can beco-introduced into DHFR-deficient CHO cells, DUKX-BII, by variousmethods including calcium phosphate coprecipitation and transfection,electroporation or protoplast fusion. DHFR expressing transformants areselected for growth in alpha media with dialyzed fetal calf serum, andsubsequently selected for amplification by growth in increasingconcentrations of MTX (e.g. sequential steps in 0.02, 0.2, 1.0 and 5 μMMTX) as described in Kaufman et al., Mol Cell Biol., 5:1750 (1983).Transformants are cloned, and biologically active BMP-15 expression ismonitored by the Rosen-modified Sampath-Reddi rat bone formation assaydescribed above in Example 3. BMP-15 protein expression should increasewith increasing levels of MTX resistance. BMP-15 polypeptides arecharacterized using standard techniques known in the art such as pulselabeling with [35S] methionine or cysteine and polyacrylamide gelelectrophoresis. Similar procedures can be followed to produce otherrelated BMP-15-related proteins.

Example 5

Biological Activity of Expressed BMP-15

To measure the biological activity of the expressed BMP-15-relatedproteins obtained in Example 4 above, the proteins are recovered fromthe cell culture and purified by isolating the BMP-15-related proteinsfrom other proteinaceous materials with which they are co-produced aswell as from other contaminants. The purified protein may be assayed inaccordance with the rat bone formation assay described in Example 3.

Purification is carried out using standard techniques known to thoseskilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGEacrylamide [Laemmli, Nature 227:680 (1970)] stained with silver [Oakley,et al. Anal. Biochem. 105:361 (1980)] and by immunoblot [Towbin, et al.Proc. Natl. Acad. Sci. USA 76:4350 (1979)]

Example 6

Using Northern analysis, BMP-15 and BMP-15-related proteins can betested for their effects on various cell lines. Suitable cell linesinclude cell lines derived from E13 mouse limb buds. After 10 days oftreatment with BMP-15 or BMP-15-related protein, the cell phenotype isexamined histologically for indications of tissue differentiation. Inaddition, Northern analysis of mRNA from BMP-15 or BMP-15-relatedprotein treated cells can be performed for various markers including oneor more of the following markers for bone, cartilage and/ortendon/ligament, as described in Table IV:

                  TABLE IV                                                        ______________________________________                                        Marker        Bone     Cartilage                                                                              Tendon/Ligament                               ______________________________________                                        Osteocalcin   +        -        -                                               Alkaline Phosphatase + - -                                                    Proteoglycan Core Protein +/-.sup.1 + +.sup.2                                 Collagen Type I + + +                                                         Collagen Type II +/-.sup.1 + +.sup.2                                          Decorin + + +                                                                 Elastin +/-.sup.3 ? +                                                       ______________________________________                                         .sup.1 Marker seen early, marker not seen as mature bone tissue forms         .sup.2 Marker depends upon site of tendon; strongest at bone interface        .sup.3 Marker seen at low levels                                         

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 17                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1541 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (B) STRAIN: PC-3                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 157..1008                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 157..633                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 634..1008                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ACATTAGTAA AGTGCTCAAT AAAATGATAA AATGCATTAA TATCATGAGC TA -            #ATTTTAGG     60                                                                 - - GCTAATTGCA ACTCTCAGTT TACATTCAGA GGTTTTCTAA GGGATGTTCA GT -            #TAAGACAC    120                                                                 - - TAATGGTCTG CCCTGTTCTT TCACATTTGT GCAGGT TCC TGG CAT - #GTA CAG ACC          174                                                                                         - #                  - #    Ser Trp His Val Gln Thr                           - #                  - #    -159            -15 - #5         - - CTG GAC TTT CCT CTA GCA TCA AAC CAG GTA GC - #A TAC GAA CTA ATC AGA          222                                                                       Leu Asp Phe Pro Leu Ala Ser Asn Gln Val Al - #a Tyr Glu Leu Ile Arg                       -150     - #           -145      - #          -140                 - - GCC ACT GTG GTT TAC CGC CAT CAA CTT CAT CT - #A GTT AAT TAC CAT CTC          270                                                                       Ala Thr Val Val Tyr Arg His Gln Leu His Le - #u Val Asn Tyr His Leu                   -135         - #       -130          - #      -125                     - - TCC TGC CAT GTG GAA ACT TGG GTT CCT AAA TG - #C CGG ACC AAG CAC TTA          318                                                                       Ser Cys His Val Glu Thr Trp Val Pro Lys Cy - #s Arg Thr Lys His Leu               -120             - #   -115              - #  -110                         - - CCT TCT TCT AAA TCG GGT TCC TCA AAG CCT TC - #T CCC ATG TCT AAA GCC          366                                                                       Pro Ser Ser Lys Ser Gly Ser Ser Lys Pro Se - #r Pro Met Ser Lys Ala          105                -100 - #                -95 - #                -90           - - TGG ACA GAG ATA GAT ATT ACA CAT TGT ATT CA - #G CAG AAG CTC TGG AAT          414                                                                       Trp Thr Glu Ile Asp Ile Thr His Cys Ile Gl - #n Gln Lys Leu Trp Asn                           -85  - #               -80  - #               -75              - - CGC AAG GGA CGG AGT GTT CTT CGC CTC CGC TT - #C ATG TGT CAG CAG CAA          462                                                                       Arg Lys Gly Arg Ser Val Leu Arg Leu Arg Ph - #e Met Cys Gln Gln Gln                       -70      - #           -65      - #           -60                  - - AAA GGC AAT GAG ACT CGT GAG TTC CGG TGG CA - #T GGC ATG ACA TCC TTG          510                                                                       Lys Gly Asn Glu Thr Arg Glu Phe Arg Trp Hi - #s Gly Met Thr Ser Leu                   -55          - #       -50          - #       -45                      - - GAT GTT GCC TTC TTG CTA CTC TAT TTC AAT GA - #C ACC GAT GAC AGA GTT          558                                                                       Asp Val Ala Phe Leu Leu Leu Tyr Phe Asn As - #p Thr Asp Asp Arg Val               -40              - #   -35              - #   -30                          - - CAG GGT AAA CTT CTT GCA AGA GGC CAA GAG GA - #G TTA ACT GAT AGG GAA          606                                                                       Gln Gly Lys Leu Leu Ala Arg Gly Gln Glu Gl - #u Leu Thr Asp Arg Glu          25                 - - #20                 - - #15                 - -        #10                                                                              - - TCT TCT TTT CTC ATG CGG AGT GTC CGC CAA GC - #A TGC AGC ATT GAA        TCT      654                                                                    Ser Ser Phe Leu Met Arg Ser Val Arg Gln Al - #a Cys Ser Ile Glu Ser                           -5 - #                  - #1               5                  - - GAT GCC TCT TGT CCT TCT CAG GAA CAT GAT GG - #G TCT GTA AAT AAC CAG          702                                                                       Asp Ala Ser Cys Pro Ser Gln Glu His Asp Gl - #y Ser Val Asn Asn Gln                    10         - #         15         - #         20                      - - TGT TCC CTC CAT CCT TAC AAG GTC AGC TTC CA - #C CAA CTA GGC TGG GAT          750                                                                       Cys Ser Leu His Pro Tyr Lys Val Ser Phe Hi - #s Gln Leu Gly Trp Asp                25             - #     30             - #     35                          - - CAC TGG ATC ATT GCT CCT CGT CTC TAT ACC CC - #A AAT TAC TGT AAA GGA          798                                                                       His Trp Ile Ile Ala Pro Arg Leu Tyr Thr Pr - #o Asn Tyr Cys Lys Gly            40                 - # 45                 - # 50                 - # 55       - - ATC TGT ACT CGG GTA TTA CCC TAT GGT CTC AA - #T TCA CCC AAC CAT GCC          846                                                                       Ile Cys Thr Arg Val Leu Pro Tyr Gly Leu As - #n Ser Pro Asn His Ala                            60 - #                 65 - #                 70              - - ATC ATT CAG AGC CTT GTC AAT GAA CTA GTG AA - #T CAC AGT GTA CCT CAG          894                                                                       Ile Ile Gln Ser Leu Val Asn Glu Leu Val As - #n His Ser Val Pro Gln                        75     - #             80     - #             85                  - - CCT TCC TGT GTC CCT TAT AAT TTT CTT CCT AT - #G AGC ATC CTC CTG ATT          942                                                                       Pro Ser Cys Val Pro Tyr Asn Phe Leu Pro Me - #t Ser Ile Leu Leu Ile                    90         - #         95         - #        100                      - - GAG ACC AAC GGG AGT ATC TTG TAC AAG GAG TA - #T GAG GGT ATG ATT GCC          990                                                                       Glu Thr Asn Gly Ser Ile Leu Tyr Lys Glu Ty - #r Glu Gly Met Ile Ala               105              - #   110              - #   115                          - - CAG TCC TGT ACA TGT AGA TAATAGTGAA GTTGTTGCTA TC - #TCAGGTTT                1038                                                                       Gln Ser Cys Thr Cys Arg                                                       120                 1 - #25                                                    - - CCCAAGAAGC TATAGATGTT TAAAGAAAAC TGTGTTAAAG CTGGCAGTGA TC -             #GAGTCGAC   1098                                                                 - - GCCCTATAGT GAGTCGTATT AGAGCTCGCG GCCGCCACCG CGGTGGAGCT CC -            #AATTCGCC   1158                                                                 - - CTATAGTGAG TCGTATTACG CGCGCTCACT GGCCGTCGTT TTACAACGTC GT -            #GACTGGGA   1218                                                                 - - AAACCCTGGC GTTACCCAAC TTAATCGCCT TGCAGCACAT CCCCCTTTCG CC -            #AGCTGGCG   1278                                                                 - - TAATAGCGAA GAGGGCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC TG -            #AATGGCGA   1338                                                                 - - ATGGAAATTG TAAGCGTTAA TATTTTGTTA AAATTCGCGT TAAATTTTTG GT -            #AAATCAGC   1398                                                                 - - TCATTTTTTA ACCAATAGGC CGAAATCGGC AAAATCCCTT ATAAATCAAA AG -            #AATAGACC   1458                                                                 - - AGATAGGGTT GGAGTGTTTG TTCCAGTTTG GGGACAAGAG TCCACTATTA AA -            #GAACGTGG   1518                                                                 - - GACTCCAACG TCAAAGGGCG AAA           - #                  - #                  1541                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 284 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Ser Trp His Val Gln Thr Leu Asp Phe Pro Le - #u Ala Ser Asn Gln Val     159             -155   - #              -150   - #              -145            - - Ala Tyr Glu Leu Ile Arg Ala Thr Val Val Ty - #r Arg His Gln Leu His                  -140     - #            -135     - #            -130               - - Leu Val Asn Tyr His Leu Ser Cys His Val Gl - #u Thr Trp Val Pro Lys              -125         - #        -120         - #        -115                   - - Cys Arg Thr Lys His Leu Pro Ser Ser Lys Se - #r Gly Ser Ser Lys Pro          -110             - #    -105             - #    -100                       - - Ser Pro Met Ser Lys Ala Trp Thr Glu Ile As - #p Ile Thr His Cys Ile     95                 - - #90                 - - #85                 - -        #80                                                                              - - Gln Gln Lys Leu Trp Asn Arg Lys Gly Arg Se - #r Val Leu Arg Leu        Arg                                                                                             -75  - #               -70  - #               -65             - - Phe Met Cys Gln Gln Gln Lys Gly Asn Glu Th - #r Arg Glu Phe Arg Trp                  -60      - #           -55      - #           -50                  - - His Gly Met Thr Ser Leu Asp Val Ala Phe Le - #u Leu Leu Tyr Phe Asn              -45          - #       -40          - #       -35                      - - Asp Thr Asp Asp Arg Val Gln Gly Lys Leu Le - #u Ala Arg Gly Gln Glu          -30              - #   -25              - #   -20                          - - Glu Leu Thr Asp Arg Glu Ser Ser Phe Leu Me - #t Arg Ser Val Arg Gln     15                 - - #10                  - #-5                  - # 1        - - Ala Cys Ser Ile Glu Ser Asp Ala Ser Cys Pr - #o Ser Gln Glu His Asp                    5    - #              10    - #              15                  - - Gly Ser Val Asn Asn Gln Cys Ser Leu His Pr - #o Tyr Lys Val Ser Phe               20         - #         25         - #         30                      - - His Gln Leu Gly Trp Asp His Trp Ile Ile Al - #a Pro Arg Leu Tyr Thr           35             - #     40             - #     45                          - - Pro Asn Tyr Cys Lys Gly Ile Cys Thr Arg Va - #l Leu Pro Tyr Gly Leu       50                 - # 55                 - # 60                 - # 65       - - Asn Ser Pro Asn His Ala Ile Ile Gln Ser Le - #u Val Asn Glu Leu Val                       70 - #                 75 - #                 80              - - Asn His Ser Val Pro Gln Pro Ser Cys Val Pr - #o Tyr Asn Phe Leu Pro                   85     - #             90     - #             95                  - - Met Ser Ile Leu Leu Ile Glu Thr Asn Gly Se - #r Ile Leu Tyr Lys Glu              100          - #       105          - #       110                      - - Tyr Glu Gly Met Ile Ala Gln Ser Cys Thr Cy - #s Arg                          115              - #   120              - #   125                          - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1609 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (B) STRAIN: hBMP-15                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 531..1376                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 531..1001                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 1002..1376                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - AAGCTGCATC TGTATAGTGA TATGACATGA GACTCTTCTT AATCCATGTA TG -             #TTCCAACA     60                                                                 - - ATTCTAAATG GACACATTAA TGGTCAACTA ATAATAATAT TGATCTTCTC CC -            #CTACATAC    120                                                                 - - AGTATGCACA CAAGATAATT CTATATTTGA GTTTTTTCCC CCGAGCCCAG CA -            #CTGTAAGT    180                                                                 - - AATCTAACAG TGAGACAGTT TCTCTTAAGA AAAACAGACT TGGGTTCAAA TC -            #TTAACTCT    240                                                                 - - ACCACATACC AGCTGTGTGT CCTTTGTCAT AGCTTCTCTG AGCCTCAATT TC -            #CTTATCTG    300                                                                 - - CAAAATGGGG ATAATAACTA TCTCATAAGA CTATTAAGAA TTAAAGAGCT AA -            #TACATGTA    360                                                                 - - AAGCATCTAG TGTATTAGTA AGTGCTCAGT AAATGATAGT ATCATTATCT TG -            #AGTTAATT    420                                                                 - - TTAGGGCTGA TTATAGCTAT CAGTCTATAT CAAGACAGTT TATGAGGAAT AT -            #TCATGTTA    480                                                                 - - AGAGGTAAGA AGCTAAACCT CTGCTCTTGT TCCCTCTTAC TTCTGCAGGT AC - #C TGG           536                                                                                        - #                  - #                  - # Thr Trp                         - #                  - #                  - # -157           - - CAT ATA CAG ATC CTG GGC TTT CCT CTC AGA CC - #A AAC CGA GGA CTA TAC          584                                                                       His Ile Gln Ile Leu Gly Phe Pro Leu Arg Pr - #o Asn Arg Gly Leu Tyr          155                -150 - #                -145 - #               -140          - - CAA CTA GTT AGA GCC ACT GTG GTT TAC CGC CA - #T CAT CTC CAA CTA ACT          632                                                                       Gln Leu Val Arg Ala Thr Val Val Tyr Arg Hi - #s His Leu Gln Leu Thr                           -135 - #               -130  - #              -125             - - CGC TTC AAT CTC TCC TGC CAT GTG GAG CCC TG - #G GTG CAG AAA AAC CCA          680                                                                       Arg Phe Asn Leu Ser Cys His Val Glu Pro Tr - #p Val Gln Lys Asn Pro                       -120     - #           -115      - #          -110                 - - ACC AAC CAC TTC CCT TCC TCA GAA GGA GAT TC - #C TCA AAA CCT TCC CTG          728                                                                       Thr Asn His Phe Pro Ser Ser Glu Gly Asp Se - #r Ser Lys Pro Ser Leu                   -105         - #       -100          - #      -95                      - - ATG TCT AAC GCT TGG AAA GAG ATG GAT ATC AC - #A CAA CTT GTT CAG CAA          776                                                                       Met Ser Asn Ala Trp Lys Glu Met Asp Ile Th - #r Gln Leu Val Gln Gln               -90              - #   -85              - #   -80                          - - AGG TTC TGG AAT AAC AAG GGA CAC AGG ATC CT - #A CGA CTC CGT TTT ATG          824                                                                       Arg Phe Trp Asn Asn Lys Gly His Arg Ile Le - #u Arg Leu Arg Phe Met          75                 - - #70                 - - #65                 - -        #60                                                                              - - TGT CAG CAG CAA AAA GAT AGT GGT GGT CTT GA - #G CTC TGG CAT GGC        ACT      872                                                                    Cys Gln Gln Gln Lys Asp Ser Gly Gly Leu Gl - #u Leu Trp His Gly Thr                          -55  - #               -50  - #               -45              - - TCA TCC TTG GAC ATT GCC TTC TTG TTA CTC TA - #T TTC AAT GAT ACT CAT          920                                                                       Ser Ser Leu Asp Ile Ala Phe Leu Leu Leu Ty - #r Phe Asn Asp Thr His                       -40      - #           -35      - #           -30                  - - AAA AGC ATT CGG AAG GCT AAA TTT CTT CCC AG - #G GGC ATG GAG GAG TTC          968                                                                       Lys Ser Ile Arg Lys Ala Lys Phe Leu Pro Ar - #g Gly Met Glu Glu Phe                   -25          - #       -20          - #       -15                      - - ATG GAA AGG GAA TCT CTT CTC CGG AGA ACC CG - #A CAA GCA GAT GGT ATC         1016                                                                       Met Glu Arg Glu Ser Leu Leu Arg Arg Thr Ar - #g Gln Ala Asp Gly Ile               -10              - #    -5              - #     1             - #  5       - - TCA GCT GAG GTT ACT GCC TCT TCC TCA AAA CA - #T AGC GGG CCT GAA AAT         1064                                                                       Ser Ala Glu Val Thr Ala Ser Ser Ser Lys Hi - #s Ser Gly Pro Glu Asn                            10 - #                 15 - #                 20              - - AAC CAG TGT TCC CTC CAC CCT TTC CAA ATC AG - #C TTC CGC CAG CTG GGT         1112                                                                       Asn Gln Cys Ser Leu His Pro Phe Gln Ile Se - #r Phe Arg Gln Leu Gly                        25     - #             30     - #             35                  - - TGG GAT CAC TGG ATC ATT GCT CCC CCT TTC TA - #C ACC CCA AAC TAC TGT         1160                                                                       Trp Asp His Trp Ile Ile Ala Pro Pro Phe Ty - #r Thr Pro Asn Tyr Cys                    40         - #         45         - #         50                      - - AAA GGA ACT TGT CTC CGA GTA CTA CGC GAT GG - #T CTC AAT TCC CCC AAT         1208                                                                       Lys Gly Thr Cys Leu Arg Val Leu Arg Asp Gl - #y Leu Asn Ser Pro Asn                55             - #     60             - #     65                          - - CAC GCC ATT ATT CAG AAC CTT ATC AAT CAG TT - #G GTG GAC CAG AGT GTC         1256                                                                       His Ala Ile Ile Gln Asn Leu Ile Asn Gln Le - #u Val Asp Gln Ser Val            70                 - # 75                 - # 80                 - # 85       - - CCC CGG CCC TCC TGT GTC CCG TAT AAG TAT GT - #T CCA ATT AGT GTC CTT         1304                                                                       Pro Arg Pro Ser Cys Val Pro Tyr Lys Tyr Va - #l Pro Ile Ser Val Leu                            90 - #                 95 - #                100              - - ATG ATT GAG GCA AAT GGG AGT ATT TTG TAC AA - #G GAG TAT GAG GGT ATG         1352                                                                       Met Ile Glu Ala Asn Gly Ser Ile Leu Tyr Ly - #s Glu Tyr Glu Gly Met                       105      - #           110      - #           115                  - - ATT GCT GAG TCT TGT ACA TGC AGA TGACAGCAAC AG - #TACGGCTA GATCAGGTTT        1406                                                                       Ile Ala Glu Ser Cys Thr Cys Arg                                                       120          - #       125                                             - - CCCAGGAAAC TGGAGGAGAG TTTAAAATAT CAGTGTTAAA GCTGCAAGTA AT -             #CCTGTACC   1466                                                                 - - AATCTGTAGG TTATATTTCT TGCCTTAAGT GTTACTTAAG TCTCTTCCCC CA -            #CTTGTGAG   1526                                                                 - - CTAGTCAGTT TATAGAAACA GTTCTGATAC CAGTCCCCTA GCATGAATCA GT -            #ACAGAGTT   1586                                                                 - - GACACTAATC AGAGCCCTTA ATG           - #                  - #                  1609                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 282 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Thr Trp His Ile Gln Ile Leu Gly Phe Pro Le - #u Arg Pro Asn Arg Gly     157     -155           - #      -150           - #      -145                    - - Leu Tyr Gln Leu Val Arg Ala Thr Val Val Ty - #r Arg His His Leu Gln          -140             - #    -135             - #    -130                       - - Leu Thr Arg Phe Asn Leu Ser Cys His Val Gl - #u Pro Trp Val Gln Lys     125                 - #-120                 - #-115                 -         #-110                                                                            - - Asn Pro Thr Asn His Phe Pro Ser Ser Glu Gl - #y Asp Ser Ser Lys        Pro                                                                                             -105 - #                -100 - #                -95           - - Ser Leu Met Ser Asn Ala Trp Lys Glu Met As - #p Ile Thr Gln Leu Val                  -90      - #           -85      - #           -80                  - - Gln Gln Arg Phe Trp Asn Asn Lys Gly His Ar - #g Ile Leu Arg Leu Arg              -75          - #       -70          - #       -65                      - - Phe Met Cys Gln Gln Gln Lys Asp Ser Gly Gl - #y Leu Glu Leu Trp His          -60              - #   -55              - #   -50                          - - Gly Thr Ser Ser Leu Asp Ile Ala Phe Leu Le - #u Leu Tyr Phe Asn Asp     45                 - - #40                 - - #35                 - -        #30                                                                              - - Thr His Lys Ser Ile Arg Lys Ala Lys Phe Le - #u Pro Arg Gly Met        Glu                                                                                             -25  - #               -20  - #               -15             - - Glu Phe Met Glu Arg Glu Ser Leu Leu Arg Ar - #g Thr Arg Gln Ala Asp                  -10      - #            -5      - #             1                  - - Gly Ile Ser Ala Glu Val Thr Ala Ser Ser Se - #r Lys His Ser Gly Pro            5            - #      10            - #      15                          - - Glu Asn Asn Gln Cys Ser Leu His Pro Phe Gl - #n Ile Ser Phe Arg Gln       20                 - # 25                 - # 30                 - # 35       - - Leu Gly Trp Asp His Trp Ile Ile Ala Pro Pr - #o Phe Tyr Thr Pro Asn                       40 - #                 45 - #                 50              - - Tyr Cys Lys Gly Thr Cys Leu Arg Val Leu Ar - #g Asp Gly Leu Asn Ser                   55     - #             60     - #             65                  - - Pro Asn His Ala Ile Ile Gln Asn Leu Ile As - #n Gln Leu Val Asp Gln               70         - #         75         - #         80                      - - Ser Val Pro Arg Pro Ser Cys Val Pro Tyr Ly - #s Tyr Val Pro Ile Ser           85             - #     90             - #     95                          - - Val Leu Met Ile Glu Ala Asn Gly Ser Ile Le - #u Tyr Lys Glu Tyr Glu      100                 1 - #05                 1 - #10                 1 -      #15                                                                              - - Gly Met Ile Ala Glu Ser Cys Thr Cys Arg                                                  120  - #               125                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Thr Arg Pro Xaa Ala Ala Xaa Ala Ala Thr Ar - #g Pro Ile Leu Glu        Xaa                                                                             1               5   - #                10  - #                15              - - Ala Ala Ala Leu Ala                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GCGGATCCTG GVANGABTGG ATHRTNGC         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ala Ser Asn His Ile Ser Ala Leu Ala Ile Le - #u Glu Xaa Ala Ala Gly      1               5   - #                10  - #                15               - - Leu Asn Thr His Arg                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GCTCTAGAGT YTGNAYNATN GCRTGRTT         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 28..99                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GGATCCTGGG AGGATTGGAT TGTGGCT CCT CGT CTC TAT ACC - #CCA AAT TAC             51                                                                                          - #            Pro Arg Leu Ty - #r Thr Pro Asn Tyr                            - #              1    - #           5                        - - TGT AAA GGA ATC TGT ACT CGG GTA TTA CCC TA - #T GGT CTC AAT TCA CCC           99                                                                       Cys Lys Gly Ile Cys Thr Arg Val Leu Pro Ty - #r Gly Leu Asn Ser Pro                10             - #     15             - #     20                          - - AACCACGCTA TAGTCCAAAC            - #                  - #                      - #119                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Pro Arg Leu Tyr Thr Pro Asn Tyr Cys Lys Gl - #y Ile Cys Thr Arg Val        1               5 - #                 10 - #                 15              - - Leu Pro Tyr Gly Leu Asn Ser Pro                                                       20                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - TCCTCGTCTC TATACCCCAA ATTACTGTAA AGGAATCTGT     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - ATCTGTACTC GGGTATTACC CTATGGTCTC AATTCACCC      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GCTTCCACCA ACTAGGCTGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CTACATGTAC AGGACTGGGC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - CATGGGCAGC TCGAG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CTGCAGGCGA GCCTGAATTC CTCGAGCCAT CATG       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - CGAGGTTAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCC TT -            #TGAAAAAC     60                                                                 - - ACGATTGC                - #                  - #                       - #          68                                                               __________________________________________________________________________

What is claimed is:
 1. An isolated DNA sequence encoding aBMP-15-related protein, said DNA sequence being isolated from abiological material selected from the group consisting of(a) λJLDc 19,which is deposited with the American Type Culture Collection (ATCC)under the Accession Number 97106; and (b) pGEMJLDc19/3.0, deposited withthe ATCC under Accession Number
 69779. 2. An isolated polynucleotidecomprising a polynucleotide which by virtue of the redundancy of thegenetic code encodes the same BMP-15-related protein expressed by thehuman DNA contained in American Type Culture Collection (ATCC) AccessionNumber 97106 or ATCC Accession Number
 69779. 3. An isolatedpolynucleotide selected from the group consisting of:(a) apolynucleotide encoding the full-length protein encoded by the DNAinsert of bacteriophage λJLDc19 deposited under ATCC Accession Number97106; (b) a polynucleotide encoding the mature protein encoded by theDNA insert of bacteriophage λJLDc19 deposited under ATCC AccessionNumber 97106; (c) a polynucleotide encoding the full-length proteinencoded by the DNA insert of subclone pGEMJLDc19/3.0 deposited underATCC Accession Number 69779; and (d) a polynucleotide encoding themature protein encoded by the DNA insert of subclone pGEMJLDc19/3.0deposited under ATCC Accession Number 69779.